Quantitative analysis of fluid samples including antigens, antibodies hormones, and other secreted proteins as well as other types of proteins frequently provides critical diagnostic data. The concept of immunoassays is well known in the art and is based on reagent impregnated capillary membranes in which an immobilized reagent in the detection zone of the strip binds to analytes conjugated with visual label such as a latex particle or metal containing compounds. In addition U.S. Pat. Nos. 3,654,090, 3,791,392 and 3,817,838 disclose enzyme labels and the means to detect such, and U.S. Pat. No. 4,062,733 discloses radioactive labels. Preferred labels are derived from metal sols. Labels such as prepared from a gold sol are bound to a ligand capable of further binding or conjugating to an analyte or other ligand or antibody. Test strips have been used in qualitative assays in a large number of applications involving analytes contained in such bodily fluids as urine and blood. Probably the earliest and most common use of assays is in detecting human chorionic gonatropin as an indicator of pregnancy in humans. These assay devices are based on test strips where the assay is performed and results read in a single step, e.g. where the liquid sample is placed onto a porous, membrane, any analyte of interest therein binds a corresponding ligand, and the results are indicated visually in a detection zone separate from the sample loading zone through formation of a specific complex. The test strips are normally contained in a dipstick or cassette allowing the user to conduct the test without any additional equipment. Such devices are disclosed in U.S. Pat. Nos. 4,231,601, 4235,601 and 5,384,264 . Assay devices that can be used with whole blood are disclosed in U.S. Pat. No. 5,821,073.
Known assay devices generally involve a sandwich assay or a competitive assay. In a sandwich assay, analyte of interest present in a fluid sample interacts with a ligand bound label or tracer resulting in a complex of analyte and label. This can occur prior to adding the analyte to the test strip or the label bound ligand can be removably deposited on the porous membrane forming the test strip. The test strip allows the label-bound analyte to move by capillary action to a sample capture zone where the analyte complex is retained by virtue of being bound to the immobilized capture ligand normally an antibody to the analyte. In the case of a gold label the presence of the analyte is indicated by color formation at the analyte test zone. Preferably the assay device also contains a second immobilized ligand band, generally identified as a control band capable of binding to label ligand even in the absence of an analyte, to show that the strip is functioning properly.
In a competitive or inhibition assay both the analyte and the label ligand are capable of reacting with the immobilized ligand in the sample capture zone and thus, both the analyte and the label bound ligand compete for attachment to the immobilized ligand in the sample capture zone. Any analyte present would displace the binding of a labeled ligand. The presence of a signal in the sample capture zone would thus indicate a negative result.
Most of the assay devices based on test strips determine the presence or absence of an analyte but do not provide but a very rough measure of the quantity of an analyte present in fluid sample. Even those that give a quantitative measurement by incorporating a standard in the test device suffer from inaccuracies resulting from temperature, air moisture, flow time variations, strip to strip variations and signal strength variations. These and other deficiencies of the prior art assay devices are overcome by the chromatographic assay device of the present invention, which provides a rapid effective, and efficient quantitative analysis of fluid samples.